Embro sac-specific genes

ABSTRACT

The present invention relates to isolated nucleotide sequences useful for the production of plants with a modified embryo sac, embryo and or endosperm development, and to transgenic cells and plants transformed with the nucleotide sequences.

This application is a divisional of co-pending U.S. application Ser. No.10/204,085 filed on Oct. 23, 2002, which is a Sec. 371 application ofPCT/EP01/02258 filed on Feb. 28, 2001, claiming priority to EuropeanApplication EP 00104366.0 filed on Mar. 2, 2000, herein incorporated byreference.

BACKGROUND OF THE INVENTION

The present invention relates to isolated nucleotide sequences usefulfor the production of plants with a modified embryo and/or endospermdevelopment, to vectors containing the nucleotide sequences, to proteinsencoded by the nucleotide sequences, to methods for obtaining thenucleotide sequences, to methods for isolating embryo sac-specific genesor proteins from a plant and to methods for producing agronomicallyinteresting plants exhibiting female sterility or allowing apomicticpropagation.

Diploid sporophytic and haploid gametophytic generations, alternate inthe life cycle of higher and lower plant species. In contrast to lowerplant species such as mosses or green algae in which the haploidgametophyte is the dominant generation, the gametophyte in higher plantspecies is dramatically reduced (Reiser and Fischer, 1993; Drews et al.,1998). Both male (pollen) and female (embryo sac) gametophytes havedeveloped from spores, the haploid products of meiosis from spores(micro- and megaspores). In angiosperms, male gametophytes (pollen) aresimple two to three-celled organisms consisting of one vegetative andone or two sperm cells, which are species-specific (Bedinger, 1992;McCormick, 1993). Three of the four megaspores in most angiospermsdegenerate and the surviving one forms the female gametophyte afterthree mitotic divisions (Reiser and Fischer, 1993; Russel, 1993). Thepredominant female gametophyte, the Polygonium type, which occurs inabout 70% of the angiosperm species (Webb and Gunning, 1990; Reiser andFischer, 1993), is deeply embedded in sporophytic tissue and consists ofonly seven cells: the egg cell, two synergids, a central cell and threeantipodals. In maize and several other species, the antipodal cellscontinue to proliferate until a group of about 20 to 40 cells is formed(Kiesselbach, 1949).

The main function of the gametophytes is to supply the gametes: male andfemale gametes fuse during fertilisation, combine their differentgenomes, and thus form a new sporohytic generation. Thus, sexualreproduction in angiosperms is initiated when pollen grains start togerminate on the female flower organ, the stigma (Cheung, 1996). Thefemale gametophyte might then function in (i) directing the pollen tubeto the ovule (Hülskamp et al., 1995; Ray et al., 1997), (ii) directingone sperm cell to the egg cell and the other to the central cell(Russel, 1992), (iii) generating a barrier to polyspermy (Faure et al.,1994; Kranz et al., 1995), (iv) preventing autonomous embryo(parthenogenesis) and endosperm development (Grossniklaus et al., 1998;Luo et al., 1999; Ohad et al., 1999) and finally (v) accumulating storesof maternal mRNAS to facilitate the rapid initiation of embryo andendosperm development after fertilisation (Dresselhaus et al., 1999b).

Morphological and structural studies of female gametophyte developmentas well as fertilisation and early embryo/endosperm development havebeen employed with many plant species (e.g. with maize: Kiesselbach,1949; Diboll 1968; Huang and Sheridan, 1994 and Arabidopsis: Webb andGunning, 1990, 1991; Muriga et al., 1993). In contrast, “The identitiesand specific functions of the haploid-expressed genes required by thefemale gametophyte are almost completely unknown” (Drews et al., 1998).This reflects the technical difficulty of identifying mutants and ofgaining access to certain developmental stages for molecular analyses.

Many mutants have been described that affect female gametophytedevelopment and function, especially in maize and Arabidopsis,suggesting that a large number of loci is essential for embryo sacdevelopment (Vollbrecht and Hake, 1995; Drews et al. 1998; Grossniklausand Schneitz, 1998. A few maternal genes functioning in the embryo sacas repressors of autonomous embryo (pathenogenesis) and/or endospermdevelopment have been recently cloned in Arabidopsis. Mea/fis1(medea/fertilisation independent seed 1) is a gametophyte maternaleffect gene probably involved in regulating cell proliferation inendosperm and partially in the embryo as well (Grossniklaus et al.,1998; Luo et al.,1999). Fis3 shows a similar phenotype and encodes aputative zinc-finger transcription factor (Luo et al., 1999): Autonomousendosperm development was observed in the fie (fertilisation independentendosperm/fis3 mutant. Mea/fis1 and fie/fis3 display homology topolycomb proteins (Grossniklaus et al. 1998; Ohad et al., 1999),proteins which are involved in long-term repression of homeotic genes inDrosophila and mammalian embryo development (Pirrotta, 1998).

At a low frequency, auxin (2, 4 D) treated sexual eggs from maize can betriggered to initiate embryo development (Kranz et al., 1995), and someegg cells initiate parthenogenetic development spontaneously. In wheat,lines have been described producing up to 90% parthenogenetic haploids(Matzk et al., 1995). The molecular mechanisms underlying theseprocesses are completely unknown. One protein (α-tubulin) was identifiedwhose expression is associated with the initiation of parthenogenesis inwheat (Matzk et al., 1997). De novo transcription from the zygoticgenome occurs relatively soon after fertilisation in maize (Sauter etal., 1998; Dresselhaus et al., 1999a), indicating that the store ofmaternal mRNA and the maternal control of embryo development is not asrelevant as it is in animal species, for example Drosophila, Xenopus orZebrafish (Orr-Weaver 1994; Newport and Kirschner 1982; Zamir et al.1997).

An important biological process linked to flower and seed development isapomixis (asexual reproduction through seeds: Koltunow et al., 1995;Vielle-Calzada et al., 1996). Due to the enormous economical potentialof apomixis once controllable in sexual crops, its application was namedafter the ‘Green Revolution’ as the ‘Asexual Revolution’ (Vielle-Calzadaet al., 1996). Up to now all approaches to isolate the ‘apomixis genes’from apomictic species failed. Genes involved in autonomous endospermdevelopment once inactivated were recently isolated from Arabidopsis(see Ohad et al., 1999; Luo et al., 1999). Autonomous embryo development(via parthenogenesis), a further component of apomixis will be necessaryto engineer the apomixis trait in sexual crops. E.g. in wheat, lineshave been described producing up to 90% parthenogenetic haploids (Matzket al., 1995). Almost no molecular data concerning parthenogenesis isavailable for higher plants: one protein (α-tubulin) was identified fromthe above described wheat lines whose expression is associated with theinitiation of parthenogenesis (Matzk et al., 1997). Nevertheless, such a‘house keeping gene’ will not be a valuable tool for genetic engineeringof the induction of parthenogenesis. Regulatory genes are needed.

Thus, from an agronomical point of view it is highly desirable toprovide plants; in particular agronomically important plants, whichallow improved hybrid breeding, apomictic propagation and/or plantshaving seedless fruits, as well as providing female sterile plants.

Thus, it is considered particularly important to develop and providemeans and methods that allow the production of plants exhibiting amodified embryo and endosperm development, in particular plantsexhibiting a modified female gametophyte development. Such plants mayprove particularly useful in commercial breeding programmes.

SUMMARY OF THE INVENTION

The technical problem underlying the present invention is to providenucleotide sequences and proteins for use in cloning and expressinggenes involved in embryo and endosperm development, in particular foruse in monocotyledonous plants which allow for the production of plantswith a modified embryo and endosperm development, in particular whichallow the production of female sterile plants or plants capable ofapomictic propagation.

The present invention solves the technical problem underlying thepresent invention by providing isolated and purified nucleotidesequences for use in cloning or expressing an embryo sac-specificnucleotide sequence selected from the group consisting of

-   -   a) the nucleotide sequence defined in any one of SEQ ID No. 1 to        8 and SEQ ID No. 13 to 31, a part or a complementary strand        thereof,    -   b) a nucleotide sequence which hybridises to the nucleotide        sequence defined in a), a part or a complementary strand        thereof,    -   c) a nucleotide sequence which is degenerated as a result of the        genetic code to the nucleotide sequence defined in a), b), a        part or a complementary strand thereof and    -   d) alleles, functional equivalents or derivatives of the        nucleotide sequence defined in a), b), c), a part or a        complementary strand thereof.

The nucleotide sequences set out in SEQ ID No. 1 to 8 and 13 to 31represent nucleotide sequences which are essential for embryo andendosperm development, and which are active in the mature embryo sac ofplants, such as maize. Thus, the present invention is inter alia basedupon the finding, isolation and characterisation of genes, in thefollowing also termed ZmES (Zea mays embryo sac) genes, which arespecifically expressed in the cells of female gametophytes of a higherplant species, in particular in the ovary or mature egg apparatus, e.g.egg cell, central cell and synergides. Expression of the genes of thepresent invention in cells outside the female gametophyte was notdetected. Furthermore, the ZmES genes of the present invention areexpressed in a temporarily specific manner, in particular theirexpression is switched off after fertilisation and expression cannot bedetected in the 2-cell or subsequent embryo stages.

The nucleotide sequence as set out in SEQ ID No. 1 to 8 and 13 to 31represent nucleotide sequences, in particular DNA sequences for use incloning or expressing an embryo sac-specific nucleotide sequence whichis essential for embryogenesis and endosperm development and is activein the embryo sac. Thus, these nucleotide sequences play a particularlyimportant role in embryogenesis and gametophyte development.Accordingly, the nucleotide sequences of the present invention areuseful for cloning, in particular isolating, embryo sac-specificnucleotide sequences, in particular regulatory elements, genetranscripts, coding sequences and/or full length genes in plants, inparticular in monocotyledonous plants. Thus, the present inventionprovides a means for the isolation of embryo sac-specific codingsequences and/or transcription regulatory elements as well as genetranscripts that direct or contribute to embryo sac-specific preferredgene expression in plants, in particular in monocotyledonous plants,such as maize.

The nucleotide sequences of the present invention are both regulatoryand protein coding nucleotide sequences.

The present invention thus relates to nucleotide sequences which areregulatory sequences, in particular transcription regulatory elementscapable of directing embryo sac-specific expression of a nucleotidesequence of interest, the regulatory sequence being selected from thegroup consisting of

-   -   a) the nucleotide sequence defined in any one of SEQ ID No. 13        to 31, a part or a complementary strand thereof,    -   b) the nucleotide sequence which hybridise to the nucleotide        sequence defined in a), a part or a complementary strand thereof        and    -   c) the alleles, functional equivalents or derivatives of the        nucleotide sequence defined in a) or b), a part or a        complementary strand thereof.

The regulatory sequences, in particular transcription regulatorysequences, are 5′ or 3′ regulatory sequences for instance promoters,transcribed, but untranslated regions (UTR) enhancers, or 3′transcription termination signals and may prove particularly useful indirecting embryo sac-specific expression of genes, in particular proteincoding sequences, of interest in plants including the protein codingsequences of the present invention. They are in particular useful fordirecting embryo sac-specific transcription of heterologous structuraland/or regulatory genes in plants, for instance DNA sequences encodingproteins modulating, inducing, repressing or suppressing embryogenesisand/or endosperm development, e.g. Mea/Fis1, Fis2, Fie/F′is3, PICKLE,LEC1 or BBM1 (Grossniklaus et al., 1998; Luo et al., 1999; Ohad et al.,1999; Ogas et al., 1999; Lotan et al., 1998; Boutilier et al.,unpublished).

Thus, the present invention provides regulatory elements such aspromoters, enhaners, UTRs and 3′ transcription termination signalsproviding for embryo sac-specific expression of a gene of interestincluding the ZmES coding sequences of the present invention. Further,regulatory elements of this specificity may be obtained by using thenucleotide sequences of the present invention to isolate in a genomicDNA library hybridising sequences encompassing further regulatoryelements.

In a particularly preferred embodiment of the present invention theabove defined promoter of the present invention is expressed in aspatially and temporally specific manner, preferably in the embryo sac.Accordingly, the proteins encoded by a gene of interest cloneddownstream from the promoter may be accumulated in embryo sacs orfruits. In a further particularly preferred embodiment, the presentinvention relates to a DNA construct with a promoter, enhancer, UTRand/or a 3′ regulatory element of the present invention operably linkedto a coding sequence for a toxic protein such as Diphteria toxin A,Exotoxin A, Barnase or RNase T1(Day et al., 1995; Koning et al., 1992;Mariani et al., 1990) specifically inhibiting the formation of embryosac tissue. The genes of interest or coding sequences of interest and/ortranscribed but untranslated regions (UTR) of interest may be cloned insense or antisense orientation to the regulatory sequences of thepresent invention.

The transcription regulatory elements of the present inventionexhibiting the above identified embryo sac-specificity, that is forinstance embryo sac-specific promoters of the present invention, may becombined to nucleotide sequences encoding proteins capable of inducingor repressing embryo genesis and/or endosperm development. Inducingembryogenesis and/or endosperm development may prove particularly usefulfor the production of plants, for example hybrid plants capable ofapomictic propagation, that is propagation without fertilisation. Theproduction of plants exhibiting a repressed and/or abortive embryoand/or endosperm development allows the production of for instancefemale sterile plants. Such plants may form sterile seed or seedlessfruit. Thus, the present invention may prove useful for all economicallyimportant plants which up until now have not been capable of apomixisand/or plants which do not provide naturally occurring female sterility.The nucleotide sequences of the present invention are useful forexpressing or suppressing an embryo sac-specific protein and/or itscoding sequence of plants such as monocotyledonous, such as maize ordicotyledonous plants such as sugar beet, including but not limited tothe proteins or coding sequences of the present invention. Thenucleotide sequences of the present invention are accordingly in aparticularly preferred embodiment useful for expressing or suppressingan embryo sac-specific protein, namely the ZmES protein or mutantvariants thereof and its target genes in plants, in particular in theembryo sac of plants. Thus, the present invention also provides a meansto allow the expression or suppression of a particular embryosac-specific or embryo sac-abundant gene in the embryo sac, therebyenabling the modification of the embryo sac and endosperm development,function and/or structure. As explained above, the present inventionthereby allows the production of plants, the embryos of which developinto plants without fertilisation and allow apomixes that is the asexualproduction of seeds.

The present invention also relates to isolated and purified nucleotidesequences which encode a protein capable of modulating embryogenesis andendosperm development, function and/or structure in plants selected fromthe group consisting of

-   -   a) the nucleotide sequence of any one of SEQ ID No. 5 to 8 and        SEQ ID No. 13 and 14, a part or a complementary strand thereof,    -   b) the nucleotide sequence encoding the amino acid sequence of        any one of SEQ ID No. 9 to 12, a part or a complementary strand        thereof,    -   c) the nucleotide sequence which hybridise to the nucleotide        sequence defined in a), b), a part or a complementary strand        thereof,    -   d) the nucleotide sequence which is degenerated as a result of        the genetic code to the nucleotide sequence defined a), b), c),        a part or a complementary strand thereof, and    -   e) the alleles, functional equivalents or derivatives of the        nucleotide sequence defined in a), b), c), d), a part or a        complementary strand thereof.

The nucleotide sequences specifically set out in SEQ ID No. 5 to 8 andSEQ ID No. 13 and 14 represent nucleotide sequences encoding a protein,in the following termed the ZmES protein, which is essential for embryoand endosperm formation. ZmES proteins are small, cysteine-rich proteinswith an N-terminal signal peptide, most likely for translocation outsidethe cell. The ZmES proteins of the present invention, namely ZmES1, 2, 3and 4 are highly homologous to each other.

The protein coding nucleotide sequences of the present invention may beuseful in engineering genetically manipulated plants exhibiting amodified embryogenesis and/or endosperm development, function and/orstructure. In particular the proteins encoded by the present nucleotidesequences may be considered to be defensins. Defensins appear to beinvolved in resistance systems against bacterial and fungal pathogens.Thus, the present invention may allow the specific modification ofplants, the embryos of which exhibit a modified resistance, inparticular improved resistance, against pathogens, for instancemicrobial pathogens. Of course, the present invention also relates toplants and methods for their production which exhibit a modifiedresistance, in particular improved resistance against pathogens comparedto a non-modified and non-transformed plant.

Plant defensins contain an N-terminal signal peptide and the maturepeptides form four disulfide bridges. This protein family includesγ-thionins, proteinase inhibitors II and P322 and other (for review seeBroekaert et al., 1995). The present invention provides a novel class ofputative plant defensins, which is specifically expressed in the femalegametophyte of maize. ZmES1-4 contains all structural components whichclassify them as plant defensins: they are small proteins, containN-terminal signal peptides and eight Cys which probably form fourintramolecular disulfide bridges, the fourth one linking the N— andC-terminal regions of the mature proteins. The predicted secondarystructure displays and α-helix and two β-stands at the same position asin the antifungal protein RsAFP1 from radish seeds, whosethree-dimensional structure has been determined by NMR spectrometry. Thesame three-dimensional structure was also determined for charybdotoxin,a neurotoxin from scorpion (Bontems et al., 1992), although this peptideis shorter at the N— and C-terminus and thus forms only three disulfidebonds. Predicted secondary and tertiary structures differ slightly, butthe positions of α-helices, β-stands and eight Cys are conserved in allplant defensins. Mature ZmES proteins are longer than most otherdefensins, but all additional amino acids are located exclusively incoil-regions, neither in α-helix nor β-stands thus allowing the samethree-dimensional structure than RsAFP1. Known plant defensins ofdiverse monocot and dicot species display higher homology among eachother than with ZmES proteins.

The protein coding nucleotide sequences or the UTRs of the presentinvention may be cloned either in sense or antisense orientation toregulatory elements, such as 5′ or 3′ regulatory nucleotide sequences,including but not limited to the regulatory nucleotide sequences of thepresent invention. Thus, using for instance antisense or cosuppressiontechnology the nucleotide sequences of the present invention, such asthe protein coding sequences, transcribed, but not translated regions(UTRs) or parts thereof, it is possible to generate plants exhibiting amodified, in particular a distorted embryogenesis and/or endospermdevelopment, function or/and structure. Such a distorted embryo genesisand/or endosperm development may cause female infertility or contributeto generating plants capable of apomixis.

Thus, the present invention also allows the modification of structure orexpression of the ZmES gene and/or protein which may lead for instanceto parthenogenetic embryo development which is an important component ofengineering the apomixis trait. For instance, the coding sequence of thepresent invention may be overexpressed in trans-formed plants due toexpression under control of a strong constitutive tissue ortissue-specific or regulated promoter. It is also possible to modify thecoding sequence of the present invention so as to allow the productionof a modified embryo sac-specific ZmES protein which in turn modifies ina desired manner embryo sac development and/or function. Mostimportantly, the present invention provides a means to specificallyinhibit the formation of a protein essential for embryo sac and/orendosperm function or development namely the ZmES protein bytransforming plants with antisense constructs comprising all or part ofthe coding sequence or, transcribed but not translated regions of theZmES gene or a part thereof in antisense orientation under the controlof its wild-type or appropriate other regulatory elements so as toeffectively bind to wild-type ZmES mRNA and inhibits its translation.Such a construct may lead upon expression to the abolishment orelimination of the wild-type ZmES function thereby producing modifiedplants.

Of course, such an eliminating effect of natural gene function may alsobe obtained using cosuppression technology. Accordingly, the nucleotidesequences of the present invention, cloned in sense orientation to atleast one regulatory element, such as a promoter into a suitable vector,are transformed into a plant, which in turn may exhibit a suppressedgene function of a wild-type ZmES gene.

The present invention also relates to processes to restore the antisenseeffect obtained by using the antisense construct mentioned above. To beable to restore the antisense effect, a further DNA construct comprisingan ZmES gene derived nucleic acid sequence in sense orientation undercontrol of a switchable or inducible promoter could be used to transformthe plant. After switching on the promoter, the antisense effect mightbe restored. An other method for restoring the above describedelimination effect is to utilise a DNA construct, in particular anantisense or co-suppression construct employing an inducible promoter tocontrol the expression of the nucleic acid sequence derived from a ZmESgene, in particular in the antisense or co-suppression construct, viaexternal factors.

In this context, it has to be understood that the antisense constructsof the present invention may not necessarily comprise all or anessential part of the coding sequence of the present invention inantisense orientation to regulatory elements, but in a particularlypreferred embodiment it is sufficient to use parts of the codingsequences or of the UTRs which are considerably shorter than the fulllength coding sequence. The length of such a sequence must be sufficientto allow effective hybridisation to the target mRNA and may be a minimumlength of 50 to 100 nucleotides.

The present invention also relates to nucleotide sequences whichhybridise, in particular under stringent conditions to the sequences setout in SEQ ID No. 1 to 8 and 13 to 31. In particular, these sequenceshave on the nucleotide level a degree of identity of ≧70% to thesequences of SEQ ID No. 1 to 8 and 13 to 31.

In the context of the present invention, nucleotide sequences whichhybridise to the specifically disclosed sequences of SEQ ID No. 1 to 8and 13 to 31 are sequences which have a degree of 60 to 70% sequenceidentity to the specifically disclosed sequence of the nucleotide level.In an even more preferred embodiment of the present invention, sequenceswhich are encompassed by the present invention are sequences which havea degree to identity of more than 70%, and even more preferred, morethan 80%, 90%, 95% and particularly 99% to the specifically disclosedsequences of the present invention on the nucleotide level.

Thus, the present invention relates to nucleotide sequences, inparticular DNA sequences which hybridise under the hybridisationcondition as described in Sambrook et al., (1989), in particular underthe following conditions, to the sequences specifically disclosed:

-   -   Hybridisation buffer: 1 M NaCl; 1% SDS; 10%    -   dextran sulphate; 100 μg/ml ssDNA    -   Hybridisation temperature: 650° C.    -   First wash: 2×SSC; 0.5% SDS at room temperature    -   Second wash: 0.2×SSC; 0.5% SDS at 65° C.

More preferably, the hybridisation conditions are chosen as describedabove, except that a hybridisation temperature and a second washtemperature of 680° C. and, even more preferred, a hybridisationtemperature and a second wash temperature of 70° C. is applied.

Thus, the present invention also comprises nucleotide sequences whichare functionally equivalent to the sequences of SEQ ID No. 1 to 8 and 13to 31, i.e. may have a different sequence but have the same oressentially the same function, in particular sequences which are atleast homologous to sequences of SEQ ID No. 1 to 8 and 13 to 31. Theinvention also relates to alleles and derivatives of the sequencesmentioned above which are defined as sequences being essentially similarto the above sequences but comprising, for instance, nucleotideexchanges, substitutions—also by unusual nucleotides—rearrangements,mutations, deletions, insertions, additions or nucleotide modificationsand are functionally equivalent to the sequences as set out in SEQ IDNo. 1 to 8 and 13 to 31.

In the context of the present invention, a number of general terms shallbe utilised as follows.

The term “promoter” refers to a sequence of DNA, usually upstream (5′)to the coding sequence of a structural gene, which controls theexpression of the coding region by providing the recognition for RNApolymerase and/or other factors required for transcription to start atthe correct site. Promoter sequences are necessary, but not alwayssufficient to drive the expression of the gene.

“Nucleotide sequence” refers to a molecule which can be single or doublestranded, composed of monomers (nucleotides) containing a sugar,phosphate and either a purine or pyrimidine. The nucleotide sequence maybe cDNA, genomic DNA, or RNA, for in-stance mRNA.

Thus, the term “nucleotide sequence” refers to a natural or syntheticpolymer of DNA or RNA which may be single or double stranded,alternatively containing synthetic, non-natural or altered nucleotidebases capable of incorporation into DNA or RNA polymers. In aparticularly preferred embodiment, the nucleotide sequence of thepresent invention is an isolated and purified nucleic acid molecule.

The term “gene” refers to a DNA sequence that codes for a specificprotein and regulatory elements controlling the expression of this DNAsequence.

The term “coding sequence” refers to that portion of a gene encoding aprotein, polypeptide, or a portion thereof, and excluding the regulatorysequences which drive the initiation or termination of transcription.The coding sequence and/or the regulatory element may be one normallyfound in the cell, in which case it is termed “autologous”, or it may beone not normally found in a cellular location, in which case it istermed “heterologous”.

A heterologous gene may also be composed of autologous elements arrangedin an order and/or orientation not normally found in the cell into whichit is transferred. A heterologous gene may be derived in whole or inpart from any source known to the art, including a bacterial or viralgenome or episome, eucaryotic nuclear or plasmid DNA, cDNA or chemicallysynthesised DNA. The structural gene may constitute an uninterruptedcoding region or it may include one or more introns bounded byappropriate splice junctions. The structural gene may be a composite ofsegments derived from different sources, naturally occurring orsynthetic.

By “operably linked” it is meant that a gene and a regulatory sequenceare connected in sense or antisense expression in such a way as topermit gene expression when the appropriate molecules (e.g.transcriptional activator proteins) are bound to the regulatorysequence.

The term “vector” refers to a recombinant DNA construct which may be aplasmid, virus, or autonomously replicating sequence, phage ornucleotide sequence, linear or circular, of a single or double strandedDNA or RNA, derived from any source, in which a number of nucleotidesequences have been joined or recombined into a unique constructionwhich is capable of introducing a promoter fragment and DNA sequence fora selected gene product in sense or antisense orientation along with anappropriate 3′ untranslated sequence into a cell.

“Plasmids” are genetic elements that are stably inherited without beinga part of the chromosome of their host cell. They may be comprised ofDNA or RNA and may be linear or circular. Plasmids code for moleculesthat ensure their replication and stable inheritance during cellreplication, and may encode products of considerable medical,agricultural and environmental importance. For example, they code fortoxins that greatly increase the virulence of pathogenic bacteria. Theycan also encode genes that confer resistance to antibiotics. Plasmidsare widely used in molecular biology as vectors to clone and expressrecombinant genes. Starting plasmids disclosed herein are eithercommercially available, publicly available, or can be constructed fromavailable plasmids by routine application of well-known, publishedprocedures. Many plasmids and other cloning and expression vectors thatcan be used in accordance with the present invention are well known andreadily available to those of skill in the art. Moreover, those of skillreadily may construct any number of other plasmids suitable for use inthe invention. The properties, construction and use of such plasmids, aswell as other vectors, in the present invention will be readily apparentto those of skill from the present disclosure.

The term “expression” as used herein is intended to describe thetranscription and/or coding of the sequence for the gene product. In theexpression, a DNA chain coding for the sequence of gene product is firsttranscribed to a complementary RNA, which is often an mRNA, and then thethus transcribed mRNA is translated into the above mentioned geneproduct if the gene product is a protein. However, expression alsoincludes the transcription of DNA inserted in antisense orientation toits regulatory elements. Expression, which is constitutive and possiblyfurther enhanced by an externally con-trolled promoter fragment, therebyproducing multiple copies of mRNA and large quantities of the selectedgene product, may also include overproduction of a gene product.

The term “suppression” refers to repression, inhibition or reduction ofendogenous gene expression.

The term “directing expression” refers to inducing, controlling,regulating, modulating, contributing or enhancing expression of anucleotide sequence.

In the context of the present invention, the term “protein” refers toany sequence length of amino acid, irrespective of its length. Thus,within the present invention the term “protein” relates to peptides,polypeptides and proteins. The protein of the present invention may bemodified by addition of carbohydrates, fats or other proteins orpeptides. The proteins of the present invention may also be modified byaddition of isotopes, amino-, acyl-, allyl-, or other groups.

The proteins of the invention that do not occur in nature are isolated.The term “isolated” as used herein, in the context of proteins, refersto a polypeptide which is unaccompanied by at least some of the materialwith which it is associated in its natural state. The isolated proteinconstitutes at least 0.5%, preferably at least 5%, more preferably atleast 25% and still more preferably at least 50% by weight of the totalprotein in a given sample. Most preferably the “isolated” protein issubstantially free of other proteins, lipids, carbohydrates or othermaterials with which it is naturally associated, and yields a singlemajor band on a non-reducing polyacrylamide gel. Substantially freemeans that the protein is at least 75%, preferably at least 85%, morepreferably at least 95% and most preferably at least 99% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically bind and recognise an analyte (antigen). The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Antibodies exist, e.g. as intactimmunoglobulins or as a number of well characterised fragments producedby digestion with various peptidases. The term “antibody”, as usedherein, also includes antibody fragments either produced by themodification of whole antibodies or those synthesised de novo usingrecombinant DNA methodologies. The term “antibody” includes intactmolecules as well as fragments thereof, such as Fab, F (ab′)₂, and Fvwhich are capable of binding the epitopic determinant. These antibodyfragments retain some ability to selectively bind with its antigen orreceptor and are defined as follows:

-   -   (1) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule can be produced        by digestion of whole antibody with the enzyme papain to yield        an intact light chain and a portion of one heavy chain;    -   (2) Fab′, the fragment of an antibody molecule, can be obtained        by treating a whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the heavy chain;        two Fab′ fragments are obtained per antibody molecule;    -   (3) (Fab′)₂, the fragment of the antibody that can be obtained        by treating a whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′)₂ is a dimer of two Fab′ fragments        held together by two disulfide bonds;    -   (4) Fv, defined as a genetically engineered fragment containing        the variable region of the light chain and the variable region        of the heavy chain expressed as two chains; and    -   (5) Single chain antibody (“SCA”), defined as a genetically        engineered molecule containing the variable region of the light        chain, the variable region of the heavy chain, linked by a        suitable polypeptide linker as a genetically fused single chain        molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988)).

The term “host cell” refers to a cell which has been geneticallymodified by transfer of a chimeric, heterologous or autologous nucleicacid sequence or its descendants still containing this sequence. Thesecells are also termed “transgenic cells”. In the case of an autologousnucleic acid sequence being transferred, the sequence will be present inthe host cell in a higher copy number than naturally occurring.

As used herein, “plant” refers to photosynthetic organisms, such aswhole plants including algae, mosses, ferns and plant-derived tissues.“Plant derived tissues” refers to differentiated and undifferentiatedtissues of a plant, including nodes, male and female flowers, fruits,pollen, pollen tubes, pollen grains, roots, shoots, shoot meristems,coleoptilar nodes, tassels, leaves, cotyledondous leaves, ovules,tubers, seeds, kernels and various forms of cells in culture, such asintact cells, protoplasts, embryos and callus tissue. Plant-derivedtissues may be in plants, or in organs, tissue or cell cultures. A“monocotyledonous plant” refers to a plant whose seeds have only onecotyledon, or organ of the embryo that stores and absorbs food. A“dicotyledonous plant” refers to a plant whose seeds have twocotyledons.

“Transformation” and “transferring” refers to methods to transfer DNAinto cells including, but not limited to, biolistic approaches such asparticle bombardment microinjection, whisker technology permeabilisingthe cell membrane with various physical (e.g., electroporation) orchemical (e.g., polyethylene glycol, PEG) treatments; the fusion ofprotoplasts or Agrobacterium tumefaciens or rhizogenes mediatedtransformation. There are no specific requirements for the plasmids usedfor the injection and electroporation of DNA in plant cells. Plasmidssuch as pUC derivatives can be used. Selectable markers are notnecessary. Depending upon the method for the introduction of desiredgenes into the plant cell, further DNA sequences may be necessary; if,for example, the Ti or Ri plasmid is used for the transformation of theplant cell, at least the right border, often, however, the right andleft border of the Ti and Ri plasmid T-DNA must be linked as flankingregion to the genes to be introduced.

If Agrobacteria are used for the transformation, the DNA to beintroduced must be cloned into specific plasmids, either into anintermediary vector or into a binary vector. The intermediary vectorscan be integrated into the Ti or Ri plasmid of the Agrobacteria due tosequences that are homologous to sequences in the T-DNA by homologousrecombination. The Ti or Ri plasmid furthermore contains the vir regionnecessary for the transfer of the T-DNA into the plant cell.Intermediary vectors cannot replicate in Agrobacteria. By means of ahelper plasmid, the intermediary vector can be transferred by means of aconjugation to Agrobacterium tumefaciens. Binary vectors can replicateboth in E.coli and in Agrobacteria, and they contain a selection markergene and a linker or polylinker framed by the right and left T-DNAborder region. They can be transformed directly into the Agrobacteria(Holsters et al., 1978). The Agrobacterium serving as a host cell shouldcontain a plasmid carrying a vir region. The Agrobacterium transformedis used for the transformation of plant cells. The use of T-DNA for thetransformation of plant cells has been extensively examined anddescribed in EP-A 120 516; Hoekema, (1985); An et al., (1985).

For the transfer of the DNA into the plant cell, plant explants can beco-cultivated with Agrobacterium tumefaciens or Agrobacteriumrhizogenes. From the infected plant material (e.g., pieces of leaf, stemsegments, roots, but also protoplasts or plant cells cultivated bysuspension) whole plants can be regenerated in a suitable medium, whichmay contain antibiotics or biocides for the selection of transformedcells.

Alternative systems for the transformation of monocotyledonous plantsare the transformation by means of electrically or chemically inducedintroduction of DNA into protoplasts, the electroporation of partiallypermeabilised cells, the microinjection of DNA into flowers, themicroinjection of DNA into micro-spores and pro-embryos, DNA transfer bywhisker technology, the introduction of DNA into germinating pollen andthe introduction of DNA into embryos by swelling (Potrykus, (1990)).

While the transformation of dicotyledonous plants via Ti plasmid vectorsystems with the help of Agrobacterium tumefaciens is well-established,more recent research work indicates that monocotyledonous plants arealso accessible for transformation by means of vectors based onAgrobacterium (Chan et al., (1993); Hiei et al., (1994); Bytebier etal., (1987); Raineri et al., (1990), Gould et al., (1991); Mooney etal., (1991); Lit et al., (1992)).

In fact, several of the above-mentioned transformation systems could beestablished for various cereals: the electroporation of tissues, thetransformation of protoplasts and the DNA transfer by particlebombardment in regenerative tissue and cells (Jähne et al., (1995)). Thetransformation of wheat has been frequently described in the literature(Maheshwari et al., (1995)) and of maize in Brettschneider et al. (1997)and Ishida et al. (1996).

In a further preferred embodiment, the invention relates to nucleotidesequences specifically hybridising to transcripts of the nucleotidesequences of the present invention. These nucleotide sequences arepreferably oligonucleotides having a length of at least 10, particularlypreferred of at least 15, most preferred of at least 50 nucleotides. Thenucleotide sequences and oligonucleotides of the present invention maybe used, for in-stance as primers for a PCR reaction or be used ascomponents of antisense constructs or of DNA molecules encoding suitableribozymes.

In a preferred embodiment of the present invention, the nucleotidesequence of the present invention is derived from dicotyledonous ormonocotyledonous plants.

In a particularly preferred embodiment of the present invention, thenucleotide sequence is derived from maize (Zea mays).

In a preferred embodiment of the present invention, the nucleotidesequence of the present invention is a DNA, cDNA or RNA molecule.

The present invention also relates to a vector comprising the nucleotidesequences according to the above, in particular to a bacterial vector,such as a plasmid or a virus.

The present invention thus also relates to vectors comprising theabove-identified nucleotide sequences in particular comprising chimericDNA constructs or non-chimeric DNA constructs such as the wild-type ZmESgene, or derivatives thereof or parts thereof. The term DNA constructrefers to a combination of at least one regulatory element and a codingsequence.

Thus, the present invention relates to recombinant nucleic acidmolecules useful in the preparation of plant cells and plants as definedabove by genetic engineering. In particular, the invention concernschimeric DNA constructs comprising a coding DNA sequence coding for awild-type ZmES protein operably linked to a promoter wherein saidpromoter is different to the promoter linked to said ZmES codingsequence in the wild-type gene i.e. either is a mutated wild-typepromoter or a promoter from another gene and/or species. In a furtherpreferred embodiment, the invention concerns chimeric DNA constructscomprising a modified coding DNA sequence coding for a mutated ZmESprotein, wherein the DNA-sequence is operably linked to a promoter whichmay be different from the promoter linked to said ZmES coding sequencein the wild-type gene or the promoter is the wild-type ZmES promoter.

Of course, the present invention also relates to chimeric antisenseconstructs comprising a DNA sequence encoding, at least partially, thenatural, that is wild-type, or modified ZmES protein, or a part thereof,which is linked to a promoter wherein said promoter is different to thepromoter linked to said ZmES coding sequences in the wild-type gene oris the wild-type promoter and wherein the orientation of the codingsequence to the promoter is vice versa to the wild-type orientation. Inone embodiment of the present invention the DNA sequence of the presentinvention used specifically to inhibit via antisense constructs thetranslation of ZmES expression from the wild-type gene is at leastpartially not derived from the ZmES coding sequence but rather containssequences from untranslated regions of the ZmES transcribed region. Boththe ZmES coding sequence and the untranslated region of the ZmES geneare also termed ZmES derived sequences. Of course the invention alsorelates to DNA constructs comprising a DNA sequence coding for thenon-chimeric wild-type ZmES protein operably linked to the wild-typepromoter. These constructs may be used to transform plant cells andplants for which the DNA construct is autologous, i.e. is the source ornatural environment for the DNA construct or for which the DNA constructis heterologous, i.e., is from another species. Plant cells and plantsobtained by using the above listed DNA constructs may be characterisedby ZmES antisense expression, multiple copies of the above DNAconstructs in their genome, that means are characterised by an increasedcopy number of the ZmES gene in the genome and/or a different locationin the genome with respect to the wild-type gene and/or the presence ofa foreign gene in their genome.

In the context of the present invention a chimeric DNA construct is thusa DNA sequence composed of different DNA fragments not naturallyoccurring in this combination. The DNA fragments combined in thechimeric DNA construct may originate from the same species or fromdifferent species. For example a DNA fragment coding for an ZmES proteinmay be operably linked to a DNA fragment representing a promoter fromanother gene of the same species that provides for an increasedexpression of the ZmES coding sequence. Preferably however, a DNAfragment coding for an ZmES protein is operably linked to a DNA fragmentcontaining a promoter from another species for instance from anotherplant species, from a fungus, yeast or from a plant virus or asynthetically produced promoter. A synthetically produced promoter iseither a promoter synthesised chemically from nucleotides de novo or ahybrid-promoter spliced together by combining two or more nucleotidesequences from synthetic or natural promoters which are not present inthe combined form in any organism. The promoter has to be functional inthe plant cell to be transformed with the chimeric DNA construct.

The promoter used in the present invention may be derived from the sameor from a different species and may provide for constitutive orregulated expression, in particular positively regulated by internal orexternal factors. External factors for the regulation of promoters arefor example light, heat, chemicals such as inorganic salts, heavy metalsor organic compounds such as organic acids, derivatives of these acids,in particular its salts.

Examples of promoters to be used in the context of the present inventionare the actin promoter from rice, the cauliflower mosaic virus (CaMV)19S or 35S promoters, nopaline synthase promoters, pathogenesis-related(PR) protein promoters, the ubiquitin promoter from maize for aconstitutive expression, the HMG (High molecular weight glutemin)promoters from wheat, promoters from Zein genes from maize, smallsubunit of ribulose bisphosphonate carboxylase (ssuRUBISCO) promoters,the 35S transcript promoter from the figworm mosaic virus (FMV 35S), theoctopine synthase promoter etc. It is preferred that the particularpromoter selected should be capable of causing sufficient expression toresult in the production of an effective amount of antisense mRNA ormodified or wild-type ZmES protein to produce flower and/or fruitmodified plants. Of course for selective expression of the ZmES proteintissue specific promoters may be used. However, in the most preferredembodiment of the present invention, i.e. the ZmES antisense constructs,the promoter may be a constitutive strong promoter, since the embryo sacspecificity of the antisense action is confined to the embryo sac due toembryo sac-specific expression of the target, i.e. the wild-type ZmESexpression.

The DNA construct of the invention may contain multiple copies of apromoter and/or multiple copies of the DNA coding sequences. In additionthe construct may include coding sequences for markers and codingsequences for other peptides such as signal or transit peptides orresistance genes for instance against virus infections or antibiotics.Useful markers are peptides providing antibiotic or drug resistance forexample resistance to phosphinstrycine, hygromycin, kanamycin, G418,gentamycin, lincomycin, methotrexate or glyphosate. These markers can beused to select cells transformed with the chimeric DNA constructs of theinvention from untransformed cells. Thus, a useful marker gene is theherbicide resistance gene Pat (phosphinotrycine acetyl transferase). Ofcourse other markers are markers coding peptidic enzymes which can beeasily detected by a visible reaction for example a colour reaction forexample luciferase, β-1,3-glucuronidase or fβ-galactosidase.

Signal or transit peptides provide the ZmES protein formed on expressionof the DNA constructs of the present invention with the ability to betransported to the desired site of action. Examples for transit peptidesof the present invention are chloroplast transit peptides ormitochondria transit peptides, especially nuclearrecognition/localisation signal peptides and endoplasmatic reticulumsignal peptides.

In chimeric DNA constructs containing coding sequences for signal ortransit peptides these sequences are usually derived from a plant, forinstance from corn, potato, Arabidopsis or tobacco. Preferably, transitpeptides and ZmES coding sequences are derived from the same plant, forinstance corn. In particular such a chimeric DNA con-struct comprises aDNA sequence coding for a wild-type ZmES protein and a DNA sequencecoding for a transit peptide operably linked to a promoter wherein saidpromoter is different to the promoter linked to said coding sequences inwild-type gene, but functional in plant cells. In particular, saidpromoter provides for higher transcription efficiency than the wild-typepromoter.

The mRNA produced by a DNA construct of the present invention mayadvantageously also contain a 5′ non-translated leader sequence. Thissequence may be derived from the promoter selected to express the geneand can be specifically modified so as to increase translation of themRNA. The 5′ non-translated regions can also be obtained from viral RNAsfrom suitable eucaryotic genes or a synthetic gene sequence.

Preferably, the coding sequence of the present invention is not onlyoperably linked to 5′ regulatory elements, such as promoters, but isadditionally linked to other regulatory elements such as enhancersand/or 3′ regulatory elements. For instance, the vectors of the presentinvention may contain functional terminator sequences such as theterminator of the octopine synthase gene from Agrobacterium tumefaciens.Further 3′ non-translated regions to be used in a chimeric construct ofthe present invention to cause the addition of polyadenylate nucleotidesto the 3′ end of the transcribed RNA are the polyadenylation signals ofthe Agrobacterium tumefaciens nopaline synthase gene (NOS) or from plantgenes like the soybean storage protein gene and the small subunit of theribulose-1,5-bisphosphonate carboxylase (ssuRUBISCO) gene. Of course,also the regulating elements of the present invention deriving from thewild-type ZmES gene may be used.

The vectors of the present invention may also possess functional unitseffecting the stabilisation of the vector in the host organism, such asbacterial replication origins. Furthermore, the chimeric DNA constructsof the present invention may also encompass introns or part of intronsinserted within or outside the coding sequence for the ZmES protein.

In a particularly preferred embodiment of the present invention, thenucleotide sequence e.g. the 5′ and/or 3′ regulatory elements of thepresent invention contained in the vector, are operably linked to anydesired gene or nucleotide sequence also termed a gene of interest,which in this context may also be a coding sequence which may be aheterologous or autologous gene. Such a gene of interest may be a gene,in particular its coding sequence, conferring for instance diseaseresistance, draught resistance, insecticide resistance, herbicideresistance, immunity and improved intake of nutrients minerals or waterfrom the soil or a modified metabolism in the plant, particularly itsembryo sac. In a particularly preferred embodiment, the vector definedabove is comprised of further regulatory elements directing or enhancingexpression of the gene of interest, such as 5′, 3′ or 5′ and 3′regulatory elements known in the art. Regulatory elements concerned inthe present invention also encompass introns or parts of intronsinserted in or outside the gene of interest. In a particularly preferredembodiment of the present invention, the regulatory element is apromoter, in particular the cauliflower mosaic virus (CaMV) 35S promoteror a promoter encoded by the nucleotide sequence selected from the groupconsisting of SEQ ID No. 13 to 31.

Thus, the nucleotide sequences of the present invention are useful sincethey enable the embryo sac-specific expression of genes of interest ofplants, in particular monocotyledonous plants. Accordingly, plants areenabled to product useful products in their embryo or endosperm. Thenucleotide sequence of the present invention may also be useful toregulate the expression of genes of interest depending upon thedevelopmental stage of the transferred cell or tissue. Furthermore, thepresent invention allows the specific modification of the metabolism inembryogenesis and endosperm development.

In a particularly preferred embodiment of the present invention, thevector furthermore contains T-DNA, in particular the left, the right orboth T-DNA borders derived from Agrobacterium tumefaciens. Of course, asequence derived from Agrobacterium rhizogenes genes may also be used.The use of T-DNA sequences in the vector of the present inventionenables the Agrobacterium mediated transformation of cells.

In a preferred embodiment of the present invention, the nucleotidesequence of the present invention, optionally operably linked toregulatory elements, is located within the T-DNA or adjacent to it.

The present invention also relates to a host cell transformed with thenucleotide sequence or the vector of the present invention in aparticular plant, yeast or bacterial cells, in particularmonocotyledonous or dicotyledonous plant cells. The present inventionalso relates to cell cultures, tissue, calluses, etc. comprising a cellac-cording to the above, for instance a transgenic cell and itsdescendants harbouring and preferably expressing the nucleotide sequenceor vector of the present invention.

Thus, the present invention relates to transgenic plant cells which weretransformed with one or several nucleotide sequences of the presentinvention as well as to transgenic plant cells originating from suchcells. Such plant cells can be distinguished from naturally occurringplant cells by the observation that they contain at least one nucleotidesequence according to the present invention which does not naturallyoccur in these cells, or by the fact that such a sequence is integratedon the genome of the cell at a location where it does not naturallyoccur, that is in another genomic region or by the observation that thecopy number of the nucleotide sequence is different, in particularhigher, than the copy number in naturally occurring plants.

Thus, the present invention also relates to trans-genic cells, alsocalled host cells, transformed with the nucleotide sequence or vector ofthe pre-sent invention, in particular plant, yeast, or bacterial cells,in particular monocotyledonous or dicotyledonous plant cells. Thepresent invention also relates to cell cultures, tissue, roots, flowers,calluses, propagation and harvest material, pollen seeds, stamen, cobs,nodes, seedlings, somatic and zygotic embryos etc. comprising a cellaccording to the above, that is, a transgenic cell being stably ortransiently transformed and being capable of expressing a nucleotidesequence of the present invention, for instance a regulatory element ora nucleotide sequence for encoding a protein modifying the embryogenesisor endosperm development of the transformed plant. The transgenic plantsof the present invention can be regenerated to whole plants according tomethods known to the person skilled in the art. The regenerated plantmay be chimeric with respect to the incorporated foreign DNA. If thecells containing the foreign DNA develop into either micro- ormacrospores, the integrated foreign DNA will in one embodiment of thepresent invention be transmitted to a sexual progeny. If the cellscontaining the foreign DNA are somatic cells of the plant, non-chimerictrans-genic cells are produced by conventional methods of vegetativepropagation either in vivo, e.g. from buds or stem cutting or in vitrofollowing established procedures known in the art.

The present invention also relates to a method of genetically modifyinga cell by transforming it with a nucleotide sequence of the presentinvention or vector according to the above whereby the ZmES1, ZmES2,ZmES3 and/or ZmES4 coding sequence or further gene of interest operablylinked to at least one regulatory element expressible in the cell,either according to the present invention or as conventionally used. Inparticular, the cell being transformed by the method of the presentinvention is a plant, bacterial or yeast cell. In a particularlypreferred embodiment of the present invention, the above method furthercomprises the regeneration of the transformed cell to a differentiatedand, in a preferred embodiment, fertile or non-fertile plant.

In a preferred embodiment of the present invention, the method totransform a cell involves direct up-take of the nucleotide sequence, inparticular by microinjection of the nucleotide sequence,electroporation, chemical treatment or particle bombardment.

The present invention also relates to a method of production of aprotein having the activity of a protein modulating embryogenesis and/orendosperm development, wherein a host cell of the present invention iscultivated under conditions allowing the synthesis of the protein, andwherein the protein is isolated from the cultivated cell and/or theculture medium. Thus, the present invention also relates to a proteinbeing preparable by a host cell of the invention or obtainable by amethod for the production of a protein of the invention.

The present invention also relates to a protein capable of modulatingembyogenesis and/or endosperm development and being encoded by thenucleotide sequences of the present invention.

The present invention also relates to derivatives of such a proteinhaving essentially the same biological activity. Such modifications maybe modifications due to amino acid substitutions, insertions, deletions,inversions, etc. Such modifications may also be constituted byglycosylation or other types of derivatisation.

The present invention also relates to an antibody or a fragment thereofwhich is reactive with a protein of the invention. These antibodies maybe used to screen expression libraries or to identify clones whichproduce the protein of the present invention. A used herein, the term“relates to an antibody” relates to detection, activation or inhibitionof molecular and cellular pathways induced by the protein of the presentinvention, in particular to modification of the embryogenesis and/orendosperm development. The term “antibody” relates to bivalent ormonovalent molecular entities that have the property of interaction withthe protein of the invention. As used herein, “antibody” refers to aprotein consisting of one or more polypeptides substantially encoded byimmunoglobulin genes or fragments of immunoglobulin genes. Light chainsare classified as either kappa or lambda. Heavy chains are classified asgamma, mu alpha, delta or epsilon which in turn define theimmunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively (fordetails see definition of the terms). The phrase “specifically bindsto”, when referring to an antibody, refers to a binding reaction whichis determinative of the presence of the domain and the presence of aheterogeneous population of proteins or other biologics. Thus, underdesignated immunoassay conditions, the specified antibody binds to aparticular domain and does not bind to a significant degree to otherproteins represented in the sample. Specific binding to the domain undersuch conditions may require an anti-body that is selected for itsspecificity for the protein of the invention. A variety of immunoassayformat may be used to select antibodies specifically immuno-reactivewith the ZmES1, ZmES2, ZmES3 and/or ZmES4 proteins. For example, solidELISA immuno-assays are routinely used to select monoclonal antibodiesspecifically immuno-reactive with the domain. The immuno-assays whichcan be used include, but are not limited to, competitive andnon-competitive assay systems using techniques such as Western blot,radioimmuno-assays, immunoprecipitation assays, precipitation reactions,gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent-immunoassays and protein A-immunoassays, to name buta few. Antibodies of the invention specifically bind to one or moreepitopes on the protein of the invention. Epitope refers to a region ofthe protein of the invention bound by an antibody, wherein the bindingprevents association of a second antibody to the protein.

In an embodiment of the invention, the antibodies are polyclonalantibodies, monoclonal antibodies and fragments thereof. Antibodyfragments encompass those fragments which interact with the protein ofthe invention. Also encompassed are chimeric antibodies typicallyproduced by recombinant methods wherein a foreign sequence comprisespart or all of an antibody which interacts with the protein of theinvention. Examples of chimeric antibodies include CDR-graftedantibodies. Also included are antibodies composed of an antibody of ananimal and a lectin of an animal or plant, in particular a lectin whichrecognises a modified carbohydrate of the membrane of cells ofembryogenesis and/or endosperm development modified plants. Antibodiesof the invention may also have a detectable label attached hereto. Sucha label may be a fluorescent (e.g. fluorscein isothiocyanate, FITC)enzymatic (e.g. horse radish oxidates) affinity (e.g. biotin) orisotopic label (e.g. ¹²⁵I). Also encompassed by the invention arehybridoma, cell lines producing a monoclonal antibody which interactwith a protein of the invention. The antibodies of the present inventionare useful in the detection of embryogenesis and/or endosperm modifieddevelopment of plants. Antibodies may be used as a part of a kit todetect the presence of the protein of the invention in a biologicalsample. Biological samples include tissue, specimens and intact cells orextracts thereof. Such kits employ antibodies having an attached labelto allow for detection. The antibodies are useful for identifyingnon-modified embryogenesis and/or endosperm development of plants.

In an preferred embodiment of the present invention, the antibody or thefragments thereof is modified, in particular used, oxidised and/oroligomerised.

The present invention also relates to a method for isolating embryosac-specific genes from a plant, whereby a preferably labelled, forinstance radioactively or fluorescently labelled, nucleotide sequence ofthe invention is used to screen gene libraries containing nucleotidesequences derived from a plant, by hybridising the gene library with thelabelled sequences of the present invention and detecting the hybridisedprobes.

The present invention also relates to a method for isolating embryosac-specific proteins from a plant, whereby an antibody of the inventionis used to screen and to isolate embryo sac-specific proteins derivedfrom the plant.

Thus, the present invention also relates to transgenic plants, parts ofa plant, plant tissue, reproductive tissue, plant seeds, plant embryos,plant seedlings, plant propagation material plant harvest material,plant leaves and plant pollen, stamen, cobs, nodes, flowers, plant rootscontaining the above identified plant cells of the present invention.These plants or plant parts are characterised by, as a minimum, thepresence of the heterologous transferred DNA construct of the presentinvention in the genome, or, in cases where the transferred nucleotidesequence is autologous to the transferred host cell, are characterisedby additional copies of the nucleotide sequence of the present inventionand/or a different location within the genome. Thus, the presentinvention also relates to plants, plant tissue, plant reproductive orvegetative tissue, plant seeds, plant seedlings, plant embryos,propagation, harvest material, leaves, nodes, cobs, stamen, fruits,flowers, pollen, roots, calluses, tassels, etc. non-biologicallytransformed which possess, stably or transiently integrated in thegenome of the cells, for instance in the cell nucleus, plastides ormitochondria, heterologous and/or autologous nucleotide sequencescontaining a) a coding sequence of the present invention and/or b) aregulatory element of the present invention recognised by thepolymerases of the cells of the said plant. In a preferred embodiment,the coding sequence of the present invention is operably linked in senseor antisense orientation to at least one regulatory element, forinstance the regulatory sequence of the present invention. In a furtherpreferred embodiment a regulatory element, in particular the regulatorysequence of the present invention is operably linked to a codingsequence of a gene of interest cloned in sense or antisense orientationto said regulatory sequence. The teaching of the present invention istherefore applicable to any plant, plant genus or plant species whereinthe regulatory elements mentioned are recognised by the polymerases ofthe cell. Thus, the present invention provides plants of many species,genera, families orders and classes, and is able to recognise theregulatory elements of the present invention or derivatives or partsthereof. Any plant is considered, in particular plants of economicinterest, for example plants grown for human or animal nutrition, plantsgrown for the contents of useful secondary metabolites, plants grown forthe content of fibres, and trees and plants of ornamental interest.Examples which do not imply any limitation as to the scope of thepresent invention are corn, wheat, barley, rice, sorghum, sugarcane,sugar beet, soybean, Brassica, sunflower, carrot, tobacco, lettuce,cucumber, tomatoes, potato, cotton, Arabidopsis, Lolium, Festuca,Dactylis or poplar.

The present invention also relates to a process, in particular amicrobiological process and/or technical process, for producing a plantor reproduction material of said plant, including an heterologous orautologous DNA construct of the present invention stably or transientlyintegrated therein, and capable of being expressed in said plants orrepro-duction material, which process comprises trans-forming cells ortissue of said plants with a DNA construct containing a nucleic acidmolecule of the present invention, i.e. a regulatory element which iscapable of causing the stable integration of the ZmES derived sequencesin particular a coding sequence in said cell or tissue and enabling thesense or antisense expression of a ZmES derived sequence, in particularcoding sequence or part thereof in said plant cell or tissue,regenerating plants or reproduction material of said plant or both fromthe plant cell or tissue transformed with said DNA construct and,optionally, biologically replicating said last mentioned plants orreproduction material or both. The present invention also relates to theabove process, wherein instead or in addition to the ZmES derived, inparticular coding sequence, a regulatory element of the ZmES gene of thepresent invention is transformed into a plant, preferably operablylinked to a coding sequence of interest.

The present invention also relates to a kit comprising the nucleotidesequence of the present invention and/or the protein of the presentinvention and/or the antibodies of the present invention. The kit of thepresent invention is useful in detecting genes involved in embryogenesisand/or endosperm development. The present invention also relates to theuse of the nucleotide sequence of the present invention and the proteinand/or the antibody of the present invention for the production ofembryo and endosperm development in modified plants.

Further preferred embodiments of the present invention are mentioned inthe subclaims.

The invention may be more fully understood from the following figuresand detailed sequence descriptions, which are part of the presentteaching. The SEQ ID No. 1 to 45 are incorporated in the presentinvention. The numbering for each DNA sequence corresponds to thegenomic clone of the gene in question.

FIG. 1 shows that mature ZmES1-4 peptides display structural homology todefensins.

(a):Homology between mature ZmES1/2 peptides, proteinase inhibitors (PI)and γ-thionins (γ Thi). N-terminal signal peptides of all proteins showno or few homology among each other and were cleaved of. The consensussequence of the putative mature peptides is shown below the alignment.Accessions of proteins used to create the alignment are as follows:putative proteinase inhibitors II and P322 from Arabidopsis thaliana,Oryza sativa, Brassica rapa, Solanum tuberosum, Glycine max (AtPI II:AC005936; OsPI: AAB17095; BrPI II: L31937; StPI P322: P20346; GmPI P322:Q07502), γ-thionins from Nicotiana tabacum, Picea abies (Nt γ Thi:P32026; Pa γ Thi: CAA62761) and an α-amylase inhibitor from Sorghumbicolor (SbAAI: S13964).

(b): The predicted secondary and tertiary structure of mature ZmESpeptides resemble the NMR structure of the plant defensin RsAFP1 fromradish and charybdotoxin, a neurotoxin from scorpion. The predicted(pred.) secondary structures are printed in grey (arrows indicateβ-strands, cylinder α-helices and lines coil regions). Tertiarystructures of mature RsAEP1 seeds (Terras et al., 1995; PDB accession#1AYJ) and charybotoxin (Bontems et al., 1992) have been determined byNMR crystallography (NMR) and are printed in black. The lines below thesequences display the position of four (RsAFPI) or three (charybdotoxin)intramolecular disulfide bridges formed between cysteine residues. Thepositions of all eight (six in the case of Charybdotoxin) Cys (C) areconserved in peptide sequences shown in (a) and (b) indicating thatprobably all plant defensins form four intramolecular disulfide bridgesand thus probably function as monomers.

FIG. 2 shows the expression of ZmES1-4 in different maize tissues, inembryo sac cells as well as in different stages of in vitro and in invivo zygotes.

(a): Multiplex RT-PCR analysis using tissues and cells indicated. Genespecific primers were used to amplify cDNA of ZmES1 and Zmcdc2,respectively. Zmcdc2 contains an intron between the primers used. Thecorresponding genomic DNA was loaded onto the last lane. Theethidiumbromide gel was blotted and hybridized with the full lengthZmES1 cDNA (below).

(b) and (c): RT-PCR analysis with cells of the female gametophyte, ofthe ovule and leaf of maize, which were manually isolated. Differentzygote and embryo stages were analysed after IVF. ZmES1 (b) or ZmES2/3/4(c) transcripts were RT-PCR amplified with gene specific primers in thecells indicated, blotted after gel separation and hybridized with thefull length ZmES4 cDNA. AP: antipodals, CC: central cell, EC: egg cell,Emb: embryo (h/d after IVF), MC: leaf mesophyll cell, Nu: nucelluscells, SY: synergid, Z: zygote (h after IVF).

FIG. 3 shows the expression of ZmES in the egg apparatus of maize.

(a) and (b): Median cut sections of ovules containing the embryo sacwere hybridized with a ZmES4 antisense probe. A purple signal shows thepresence of ZmES4 clearly in the synergids and more faint in the egg andcentral cell. In nucellus and integuments no signal was detected. (c): Asimilar section was hybridized with a ZmES4 sense probe, showing nohybridization signal.

(d): A median cut section of an ovule containing the embryo sac wasstained with acridine orange to show nuclei and to monitor RNA contentof sections used for in situ hybridization. CC: central cell, EC: eggcell, SY: synergid. Bars: 60 μm.

FIG. 4 is a whole mount in situ hybridization showing that ZznEStranscripts are uniformly distributed in the cytoplasm of isolatedfemale gametophyte cells.

(a): Egg and nucellus cells hybridized with a ZmES4 antisense probe.

(b): Egg cell hybridized with a ZmES4 sense probe.

(c): Central cell, synergid and nucellus cells hybridized with a ZmES4antisense probe.

(d): Central cell and nudellus cells hybridized with a ZmES4 senseprobe.

(e), (f) and (g): Acridine orange staining to display total RNAdistribution within synergid, central cell and nucellus cells,respectively. Bars: 50 μm.

FIG. 5 shows green fluorescence protein (GFP) expression in transgenicmaize plants driven by 1594 bp promoter region upstream of thetranscription start point of ZmES4.

(a): The expression pattern of ZmES4::GFP fusion protein in the ovarytissue around the embryo sac under light microscopy.

(b): The same preparation as in (a) under UV light microscopy.

(c): The same preparation as in (a) using CLSM.

SEQ ID No. 1 represents the full length cDNA sequence of the ZmES1 (Zeamaysembryo sac) gene, from and including position 619 towards the 5′end, up to and including position 1204.

SEQ ID No. 2 represents the full length cDNA sequence of the ZmES2 gene,from and including position 1 towards the 5′ end, up to and, includingposition 517.

SEQ ID No. 3 represents the full length cDNA-sequence of the ZmES3 gene,from and including position 1 towards the 5′ end, up to and includingposition 501.

SEQ ID No. 4 represents the full length cDNA sequence of the ZmES4 gene,from and including position 1850 towards the 5′ end, up to and includingposition 2430.

SEQ ID No. 5 represents the protein coding region of ZmES1, from andincluding position 702 towards the 5′ end, up to and including position977 (excluding the stop-codon).

SEQ ID No. 6 represents the protein coding cDNA region of ZmES2, fromand including position 77 towards the 5′ end, up to and includingposition 349 (excluding the stop-codon).

SEQ ID No. 7 represents the protein coding cDNA region of ZmES3, fromand including position 78 towards the 5′ end, up to and includingposition 350 (excluding the stop-codon).

SEQ ID No. 8 represents the protein coding region of ZmES4, from andincluding position 1927 towards the 5′ end, up to and including position2199 (excluding the stop-codon).

SEQ ID No. 9 represents the amino acid sequence of the ZmES1 protein.

SEQ ID No. 10 represents the amino acid sequence of the ZmES2 protein.

SEQ ID No. 11 represents the amino acid sequence of the ZmES3 protein.

SEQ ID No. 12 represents the amino acid sequence of the ZmES4 protein.

SEQ ID No. 13 represents the full length genomic clone of the ZmES1gene, from and including position 1 towards the 5′ end, up to andincluding position 1204; at the position 587 is a TATA sequence, at theposition 702 is a ATG sequence (start codon) at the position 978 is aTAA Sequence (stop codon).

SEQ ID No. 14 represents the full length genomic clone of the ZmES4gene, from and including position 1 towards the 5′ end, up to andincluding position 2430; at the position 1817 is a TATA sequence, at theposition 1927 is a ATG sequence (start codon) and at the position 2200is a TGA sequence (stop codon).

SEQ ID No. 15 represents the full length promoter of the ZmES1 gene,from and including DNA sequence of the position 1 towards the 5′ end, upto and including position 701.

SEQ ID No. 16 represents a partial DNA sequence of the promoter of theZmES1 gene; the Sequence spans the region from and including position501 towards the 5′ end, up to and including position 701.

SEQ ID No. 17 represents a partial DNA sequence of the promoter of theZmES1 gene; the sequence spans the region from and including position201 towards the 5′ end, up to and including position 701.

SEQ ID No. 18 represents the transcribed 5′-untranslated region (UTR) ofthe ZmES1 gene, from and including position 619 towards the 5′ end, upto and including position 701.

SEQ ID No. 19 represents the transcribed 5′-untranslated region of theZmES2 gene, from and including position 1 towards the 5′ end, up to andincluding position 76.

SEQ ID No. 20 represents the transcribed 5′-untranslated region of theZmES3 gene, from and including position 1 towards the 5′ end up to andincluding position 77,

SEQ ID No. 21 represents the transcribed 5′-untranslated region of theZmES4 gene, from and including position 1850 towards the 5′ end, up toand including position 1926.

SEQ ID No. 22 represents the 3′- termination region including the Poly Aaddition sequence of the ZmES1 gene, from and including position 978towards the 5′ end, up to and including position 1223.

SEQ ID No. 23 represents the 3′-termination region including the Poly Aaddition sequence of the ZmES2 gene, from and including position 350towards the 5′ end, up to and including position 537.

SEQ ID No. 24 represents the 3′-termination region including the Poly Aaddition sequence of the ZmES3 gene, from and including position 351towards the 5′ end, up to and including position 519.

SEQ ID No. 25 represents the 3′-termination region including the Poly Aaddition sequence of the ZmES4 gene, from and including position 2200towards the 5′ end, up to and including position 2449.

SEQ ID No. 26 represents the full length DNA sequence of the promoter ofthe ZmES4 gene, from and including position 1 towards the 5′ end, up toand including position 1926.

SEQ ID No. 27 represents a partial DNA sequence of the promoter of theZmES4 gene; the sequence spans the region from and including position1699 toward the 5′ end, up to and including position 1926.

SEQ ID No. 28 represents a partial DNA sequence of the ZmES4 gene; thesequence spans the region from and including position 1499 towards the5′ end, up to and including position 1926.

SEQ ID No. 29 represents the partial DNA sequence of the promoter of theZmES4 gene; the sequence spans the region from and including position999 towards the 5′ end, up to and including position 1926.

SEQ ID No. 30 represents the partial DNA sequence of the promoter of theZmES4 gene; the sequence spans the region from and including position499 towards the 5′ end, up to and including position 1926.

SEQ ID No. 31 represents the partial DNA sequence of the promoter of theZmES4 gene; the sequence spans the region from and including position199 towards the 5′ end, up to and including position 1926.

SEQ ID No. 32 to 44 represent primers used in obtaining the ZmES genes.

SEQ ID No. 45 represents a 1594 bp promotor region of ZmES4 that wasused for monitoring expression of the promotor of ZmES4 after stableintegration into the maize genome.

The following examples are offered to more fully illustrate theinvention, but are not construed as limiting the scope thereafter.

EXAMPLES

Materials and Methods Used Throughout the Examples

Plant material, isolation of cells from the embryo sac, in vivo and invitro fertilisation Maize (Zea mays) inbred line A188 (Green andPhillips, 1975) were grown under standard green house conditions.

Cells of the embryo sac were mechanically isolated from digested ovuletissues with glass needles and transferred using a hydraulicmicrocapillary system according to Kranz et al. (1991). In vitro zygoteswere generated after fusing isolated gametes by a short electric pulseand cultivated as described (Kranz and Lörz, 1993). In vivo zygotes weregenerated as described by Cordts et al. (2001). The cells were collectedand fixed on glass slides or stored in 200 nl each at −80° until usage.

Light microscopy 2 mm thick slices of spikelets were fixed in 4%paraformaldehyde in 0.005 M phosphate buffer, pH 7.2. The slices werewashed in 0.1 M phosphate buffer, dehydrated in ethanol series andinfiltrated in gradient steps of butylmethyl methacrylate, followed byUV polymerisation (Wittich and Vreugdenhil, 1998). Sections of 3 pm weremade with a Reichert Ultramicrotome, stretched on water, and dried onmicroscope slides at 600 C for 1 hour. The resin was removed from thesections by washing the slides in pure acetone for 15 minutes. Theslides were then washed in water and sections were stained withtoluidine blue (O'Brien et al., 1965).

Differential Plaque- and Reverse Northern Screening

RT-PCR-based cDNA libraries generated from isolated egg cells and invitro zygotes (Dresselhaus et al., 1994; 1996) were screened bydifferential plaque screening (Dresselhaus et al., 1996). Double plaquelifts were made from 15 cm plates of the egg cell library at a densityof 500 p.f.u. (plaque-forming units). The filters were hybridised eitherwith PCR amplified [₃₂P]-cDNA from the egg cell or the zygote cDNAlibrary. CDNA clones from the egg cell library selected by thisscreening were further analysed by a differential insert screening(“reverse Northern screening”; Dresselhaus et al., 1999 alb). The cDNAclones were amplified by PCR, separated in agarose gels, blotted andhybridised either with the radiolabelled, PCR amplified cDNA populationof the egg cell library or the zygote library. The isolated cDNA cloneswere further hybridised, with uncloned cDNA populations of egg cells andzygotes as a control. The following gene specific primers were used tospecifically amplify the different subgroups of the ZmES gene family:ZmES1 (5′-CCCTTGGATTGGATTGGATCG-3′ SEQ ID No. 32 and5′-ACCACCGGTTTCCTGCTGTC-3′ SEQ ID No.33) and ZmES2/3/4(5′-TCTTCACGAGGGAAGCTGTCT-3′ SEQ ID No. 34 and5′-GCACTGCACCCACCGCTCTT-3′ SEQ ID No. 35). RT-PCR

Total RNA from different maize tissues was isolated using TRIZOL®(Gibco-BRL) after the manufacturers recommendations. For quantification,total RNA was separated in a formaldehydgel, transferred over-night with10×SSC to Hybond N⁺ membrans (Amersham Pharmacia Biotech) and hybridizedwith a radio-labelled 18S rDNA probe. RNA was quantified using abioimager system (BAS-1000, Fuji). One μg quantified total RNA of eachsample was used for RT-PCR analysis. To avoid ampification from remnantgenomic DNA in the sample, total RNA was treated prior RT reaction withDNaseI for 15 min at RT after the manufacturers recommendations(Gibco-BRL). The reaction was stopped by adding EDTA (25 mM) and byincubation for 10 min at 70° C.

The RNA was primed with a T¹⁴- A/G/C-primer (Metabion) and re-versetranscribed in 20 μl final volume using 50 U Superscript™ reversetranscriptase (Gibco-BRL) for 60 min. The reaction was stopped byincubating for 10 min at 70° C. Multiplex RT-PCR: 50 μl reactionscontaining 100 ng of total RNA and the primer pairs for ZmES1 (forward:5′ CCCTTGGATTGGATTGGATCG-3′ SEQ ID No.32, reverse:5′-GTCATTACCACCACAGACTTC-3′ SEQ ID No. 42) and Zmcdc2 (forward:5′-ACTCATGAGGTAGTGACATT-3′ SEQ ID No.43, reverse:5′-CATTTAGCAGGTCACTGTAC-3′ SEQ ID No. 44; Sauter et al., 1998) were runon a TGradient Cycler (Biometra). 30 cycles, with a first denaturationstep at 96° C. for 60 sec, were performed with the following parameters:96° C. for 30 sec, 58° C. for 30 sec and 72° C. for 60 sec, followed bya final extension at 72° C. for 10 min before soaking at 4° C.Single-cell RT-PCR analysis with one primer pair was carried out asdescribed by Richert et al. (1996) using primers SEQ ID No.32-35 with afew modifications as described by Cordts et. (2001).

DNA Gel Blot Analyses

Extraction of genomic DNA from 10-day old seedlings was performedaccording to Dellaporta et al. (1983). 10 μg genomic DNA was digestedwith the restriction enzymes indicated and resolved on 0.8% agarosegels. DNA was transferred to Hybond N⁺ membranes (Amersham PharmaciaBiotech) with 0.4 M NaOH. Blots were hybridised with radioactive probesprepared by Prime-It Random Primer Labelling Kit (Stratagene, USA) inCHURCH buffer (7% SDS, 0.5 M NaH₂PO₄, pH 7.2, 1 mM EDTA) containing 100μg/ml salmon sperm DNA. Filters were washed with de-creasingconcentrations of SSC, with a final wash at 65° C. in 0.2×SSC/0.1% SDS.Filters were exposed at −70° C. to Kodak X-Omat AR films usingintensifier screens.

In situ hybridization Ovule pieces containing embryo sacs were fixed in4% formaldehyde, 0.25% glutaraldehyd and embedded inbutyl-methyl-methacrylat (BMM) (Gubler 1989; Baskin et al. 1992). Theembedded tissues were sectioned on glass knives with an ultramicrotom at5 to 7 pm thickness.

A whole mount in situ hybridisation protocol was developed for isolatedcells of the embryo sac. Cells were temporarily collected afterisolation in 540-650 mosmol kg⁻¹ mannitol and then placed in drops offixation solution (540-650 mosmol kg⁻¹ mannitol, 4% formaldehyde, 0.25glutaraldehyd) on mounted glass coverslides (bindsilane; Wacker-Chemie).The cells were always submerged in liquids. After 30 min incubation, thesamples were postfixed for 15 min by adding droplets of PBS-buffercontaining 20% acetic acid. The samples were dehydrated by passagethrough a graded ethanol series (10% to 70%) and stored at 4° C. ordirectly used for further steps. The solution was gradually substitutedwith hybridisation solution (10 mM Tris-HC1 (pH 7,5), 300 mM NaCl, 50%formamid, 1 mM EDTA, 1×Denharts and 10% dextransulphate) or inDig-Easy-Hyb (Boehringer Mannheim) containing 250 ng/ml tRNA and 100μg/ml poly(A) oligonucleotide. The glass cover slides with stickingcells were placed in small (diameter of 35 mm) plastic petri dishes in avolume of 500 pμ hybridisation solution. 1 μg/ml labelled probe wasadded to the hybridisation solution. Washing and detection steps weremade by submerging the plastic dishes in larger volumes of theappropriate solutions. Hybridisation, washing steps and detection wereperformed for sectioned material and whole mount cells in the samemanner.

Antisense and sense RNA probes were labelled in vitro from cDNA insertsin pBluescript II SK—with digoxigenin-UTP by T7 or T3 RNA polymeraseusing a digoxygenin RNA Labelling kit (Boehringer Mannheim).Hybridisation was carried out at 43° C. overnight. Washing steps wereperformed as follows: 10 min at 43° C., 30 min in 1×SSC/0.01% SDS andonce 30 min in 0.5×SSC/0.01% SDS followed by digestion with RNase A(Boehringer, Mannheim). After washing three times in 1% NaCl, detectionwas made using an anti-digoxigenin antibody conjugated with alkalinephosphatase and NBT/BCIP detection system (Boehringer Mannheim).

DNA and Protein Sequence Analyses

Selected cDNAs were excised from the λXZAP XR vector according to themanufacturer's specifications (Stratagene). All clones were sequencedfrom both directions using Taq DNA polymerase FS Cycle Sequencing Kit(PE APPLIED BIOSYSTEMS) and the 373A and 377 automated DNA sequences(APPLIED BIOSYSTEMS). DNA and amino acid sequence data were furtherprocessed using the PC DNASIS program software package (Hitachi SoftwareEngineering). Sequence data were compiled and compared online with EMBL,GenBank, DDBJ, SwissProt, PIR and PRF data bases with FASTA and BLASTalgorithms (Pearson,1990). Protein alignment was performed with theCLUSTAL W program (Thompson et al., 1994). Prediction of proteinlocalization sites was performed online using PSORT(http://psort..nibb.ac.jp) and the signal peptide cleavage site wasidentified after Nielsen et al. (1997). Secondary and tertiary structureprediction was performed online at http://insulin.bio.warwick.ac.uk andwith PDB (protein data bank) at http://pdb.ccdc.cam.ac.uk. Prediction ofprotein localization sites was performed online using the PSORT IIsoftware package (Nakai, K. and Horton, Trends Biochem. Sci, 24(1) 34-35(1999)), and the signal peptide cleavage site was identified afterNielsen et al. (1997). Secondary and tertiary structure prediction wasperformed with the PSIPRED server (McGuffin, et. al, Bioinformatics, 16;pp. 404-05) and with PDB(Protein Data Bank).

Isolation of Genomic Clones

Genomic DNA was isolated from the maize inbred line A188 according toDellaporta et al. (1983), partially digested with Sau3A and sizefractionated using a saccharose gradient (Sambrook et al., 1989). DNAfragments between 13-23 kb were cloned into the BamHI site of the LambdaDash II vector (Stratagene) according to the manufacturersspecifications. Genomic clones containing ZmES1-4 sequences wereidentified after using ZmES1-4 cDNA clones as probes.

In order to obtain upstream sequences, the Universal Genome Walker Kit(Clontech) was used. The protocol from the kit was modified as follows:to prepare adaptor ligated DNA, 2,5 pμ of λ-DNA was digested in 100 μlreaction volumes with 80 U of different restriction enzymes (Oral,EcoRV, Pvull, Scal and Stul) overnight at 37° C. using buffersrecommended by Clontech. The DNA was extracted once withchloroform/isoamyl alcohol (24:1) vol./vol., once with chloroform, andthen precipitated by addition of 1/10 (vol/vol) 3 M NaOAc (pH 4.5), 20μg glycogen and 2 vol. of 95% EtOH. After vortexing, the tubes wereimmediately centrifuged at 15,000 rpm in a microcentrifuge for 5 min.

The pellets were washed with 80% EtOH and immediately centrifuged asabove for 5 min, air dried and dissolved in 20 μl of 10 mM Tris-HCl (pH7.5), 0.1 mM EDTA. From each tube 4 μl of DNA was ligated to an excessof adaptor overnight at 16° C. under the following conditions: 1.9 μlGenome Walker Adaptor (25 μM), 0.5 μl T4 DNA Ligase (1 U/μl), 1.6 μl5×ligation buffer in a total volume of 8 μl. The ligation re-action wasterminated by incubation of the tubes at 70° C. for 5 min, then diluted10-fold by addition of 72 μl of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA(pH 7.4). The Biometra trioblock was used for all incubation reactions.PCR amplifications were performed using TaqDNA Polymerase (Gibco Lifetechnologies). Primary PCR reactions were conducted in 50 μl volumecontaining 1 μl of ligated and diluted DNA, 5 μl 10×PCR buffer, 1 μldNTP (10 mM each), 2.2 μl Mg(OAc)₂(25 mM), 1 ml adaptor primer (10 mM)API (5′-GTAATACGACTCACTATAGGGC-3′, SEQ ID No. 38) and each 1 μl genespecific primer (10 mM) GSP1 (5′-CTTGACGCAGTAGCAGAGAATCCCGTC-3′, SEQ IDNo. 37) or GSP2 (5′-CAGTAGTCCGACCGCACGCACAG(A/g) TG-3′, SEQ ID No. 36),and 1.25 U Taq DNA Polymerase. The PCR cycles were conducted asdescribed by the manufacturer. A secondary PCR (nested PCR) reaction wasperformed with 1 μl of a 100 fold dilution of the primary PCR usingadaptor primer AP2(5′-ACTATAGGGCACGCGTGGT-3′, SEQ ID No. 39) and nestedGSP3(5′-CAGACAGCTTCCCTCGTGAAGCTCCCATTG-3′, SEQ ID No.40)and GSP4(5′-TCTGc/T)GTCAGGCAGTC(T/g)CGTGCCTCAAC-3′, SEQ ID No.41), respectively. The PCRcycles of the second reaction were conducted as described by themanufacturer. PCR products were cloned using TA cloning vectors(Invitrogen) and sequenced. Upstream sequences of ZmES1 and 4 could thusbe cloned. The analysis of the genomic clones and genomic DNA furthershowed that ZmES1-4 gens contain no introns.

Biolistic Transformation and Analyses of Transgenic Maize Plants

1594 bp promoter region of ZmES4 (SEQ ID No. 45) was used for monitoringexpression of the promoter of ZmES4 after stable integration into themaize genome. A construct consisting of SEQ ID No. 45, a part of thecDNA of ZmES4 (bp 2 to bp 351 of SEQ ID No. 4), the coding region of GFP(pMon30049; Monsanto) and the NOS-terminator (McElroy et al., 1995) wasgenerated using the vector Litmus 29 (New England Biolabs). Immatureembryos from maize inbred line A188 were isolated 12 days after handpollination and co-bombarded with the construct described above and thep35S::pat vector (P. Eckes, Aventis, unpublished) containingphosphinotricinacetyl-transferase as selection marker. Experimentalprocedures followed the protocol of Brettschneider et. al. (1997),except that embryos were bombarded with partial vacuum 28 inch Hg andgas pressure 1350 psi. Cultivation, regeneration and selection wascarried out as described by Brettschneider et. al. (1997).

GFP Analysis in Transgenic Maize Plants

Immature ears with silks of 15 cm length (counted from bottom part ofthe ear), were harvested from transgenic plants (lines containing fulllength integrations of the pZMES::ZMES::GFP::NOS construct as analysedby gel blots; data not shown), kernel were excised and cut in the middlepart with razor blades or scalpels. The part of kernels containing theembryo sac was transferred into a 650 mOsm mannitol solution and thenucellar tissue dissected out of the submerged ovary tips. The embryosac was preparated with fine-tipped glass needles using an invertedmicroscope. The preparations were analysed by light and UV microscopy,or a Confocal Laser Scanning Microscope (CLSM) for presence offluorescence in ovary tissues.

Example 1

Isolation of the ZmES Gene Family from Maize Egg Cells

The female gametophyte of maize is deeply embedded in the maternaltissues of the ovule. Gene expression patterns in cDNA libraries ofunfertilised egg cells and in vitro zygotes were compared as a startingpoint for molecular investigations. With the aim of identifying genescompletely downregulated after IVF (in vitro fertilisation) and notexpressed elsewhere in the plant, 29,000 pfu (plaque forming units) fromthe egg cell library were analysed. Double plaque lifts were hybridizedwith the egg cell cDNA population and either with cDNA from in vitrozygotes or cDNA from seedlings. 250 clones were selected and furtheranalysed in reverse Northern blot analysis. 44 different cDNA clones,which were highly represented in the egg cell library and not or weaklyin the zygote library, were fully sequenced. Ten cDNAs were highlyhomologous to each other and were further analysed. These ten cDNAsrepresent four different genes (ZmES1-4) (see SEQ ID Nos. 1 to 4).

A reverse Northern blot indicates that the whole gene family iscompletely switched off after IVF and minimal transcript amounts remaindetectable 18 h after IVF.

DNA and protein sequence alignments display a high degree of sequencehomology among the different ZmES gene family members; even 5′ and 3′UTRs (untranslated regions) (SEQ ID No. 18 to 25) are highly conserved.ZmES1 is more distinct from the other ZmES members, both at the DNA andprotein level, but all general features, such as transcription startpoint, two stop codons, putative CPE element and poly(A) signal site atthe DNA level, are identical. At the protein level, signal peptidecleavage site and cysteine residues are also identical. The longest cDNAclones of all ZmES members start more or less at the same position withone or two Gs. These Gs are missing in genomic clones of ZmES1 (SEQ IDNo. 13) and ZmES4 (SEQ ID No. 14) and most likely result from the ^(7m)Gcap at the 5′ end of all messenger RNAs. This is a strong indicationthat the isolated cDNAs with SEQ ID No. 1 to 4 are of full length.

ZmESI-4 encode small proteins of 92 and 91 aa (amino acids), shown inSEQ ID No. 9 to 12, respectively. A putative signal peptide is locatedat the N-terminus of all proteins. A hydrophobic amino acid cluster atthe N-terminus of the precursor protein is framed by two basic and onebasic amino acid, respectively. The predicted cleavage site is afterposition 31 (ZmES1) (SEQ ID No. 9) or 30 (ZmES2/3/4) (SEQ ID Nos.10-12). It is further predicted that the proteins are translocatedoutside the cell, including the cell wall. The MW of ZrnESI-4 precursorproteins is 9.7 kDa, and the pI varies between 8.1 and 8.5, and is thusslightly basic. The mature proteins are cysteine-rich and extremelyconserved, with little variation at the C-terminus and the predicted MWis 6.5 kDa, while the pI is between 7.9 and 8.3. The structural homologyto defensins is shown in FIG. 1.

To investigate the size of the ZmES gene family and their presence inother related genomes, genomic Southern blot analysis with two differentmaize inbred lines and the diploid maize relative Tripsacum dactyl aideswas performed. All enzymes used do not cut within the cDNA, nor withinthe corresponding genomic sequences. Four bands were detected in A188,the maize line used to generate the cDNA libraries. The same number ofbands was detected in another inbred line (B73), while two to threebands were detected in Tripsacum. According to the present invention,the whole gene family from the maize inbred line A188 was isolated.

SEQ ID Nos. 1 to 4 illustrate the full length cDNA sequences, SEQ IDNas. 5 to 8 the protein coding nucleotide sequences and SEQ ID Nos. 9 to12 the amino acid sequence of ZmES 1 to 4.

SEQ ID Nos. 13 and 14 represent the full length nucleotide sequences ofthe genomic clone of ZmES1 and 4, thus incorporating in 5′ to 3′direction the promoter, the transcribed 5′ untranslated region (UTR),the protein coding region and the 3′ transcription termination region.SEQ ID Nos. 15 to 17 represent the full length promoter and promoters ofreduced length of the ZmES1 gene. SEQ ID Nos. 18 to 21 representtranscribed, but not translated regions of ZmES1 to 4 possiblyfunctioning as expression modulating elements. The UTR nucleotidesequence elements are included in most of the promoter fragmentsillustrated in SEQ ID Nos. 13 to 31. However, the present invention alsoencompasses the promoters and fragments thereof indicated in SEQ ID Nos.13 to 31 wherein the UTRs of SEQ ID Nos. 18 to 21 are missing.

SEQ ID Nos. 22 to 25 represent the 3′ transcription terminationsequences of ZmES1 to 4 containing possibly important elements for theregulation of transcription.

SEQ ID Nos. 26 to 31 represent full length promoters and promoters ofreduced length capable of promoting and/or enhancing transcription withembryo sac-specificity.

Example 2

ZmES1-4 are Specifically Expressed in All Cells of the Embryo Sac andSwitched Off After IVF

In order to investigate whether ZmES1-4 are exclusively expressed in eggcells, total RNA, poly(A)⁺ RNA Northern blot and RT-PCR analysis wasperformed with many different tissues at distinct developmental stages.No signal was obtained in any tissue tested (see FIG. 2 as an example).

Tissue in situ hybridisation was performed to investigate the expressionof ZmES1-4 in ovules at maturity; strong signals were detected in thecytoplasm of two synergides already after short detection time (see FIG.3). Signals in nucellus cells, integuments or ovary tissues were neverobserved. A problem with in situ hybridisation of ovule tissue was thatthe structure after embedding in paraffin wax and sectioning was notconserved. Tissues had to therefore be embedded in BMM(butylmethyl-methacrylate), which however only allowed the generation ofvery thin sections. The structure after BMM embedding is conserved, thesections still contain RNA, but cells contain only few cytoplasm due tothe slight thickness of each section, thus making in situ hybridisationless efficient. In addition, all embryo sac cells are very large andhighly vacuolated, thus making the detection of transcripts within thesecells even more difficult. The embryo sac in its different cell typeswas therefore dissected and single cell RT-PCR was applied toinvestigate ZmES1-4 transcript contents.

As shown in FIG. 2 ZmES1-4 transcripts are expressed at comparablelevels in all egg cells tested and in most of the synergides and centralcells. Some 15 antipodal cells were used under the same RT-PCRconditions for a single reaction, and a much smaller signal wasdetected, or no signal at all. After IVF, ZmES2/3/4 transcripts weredetectable at very low levels in few zygotes up to 42 h after IVF. ZmES1transcript was detected until 18 h after IVF and 24 h after in vitropollination in in vivo zygotes. After the first cell division, whichgenerally occurs between 42 and 46 h after IVF, transcripts were nolonger detectable. No transcripts could be detected in different embryostages, in nucellus or leaf mesophyll cells.

Example 3

ZmES1-4 Transcripts are Uniformly Distributed in Cytoplasms of EmbryoSac Cells

An in situ hybridisation protocol with isolated embryo sac cells wasdeveloped to investigate whether ZmES1-4 transcripts are localized atdifferent poles within the cells of the embryo sac: as shown in FIG. 4transcripts were detected in egg cells, synergides and central cells. Nodetection was observed in nucellus cells adjacent to central cells. Itseems that ZmES1-4 transcripts are uniformly distributed in these cells,which is best seen in egg cells: in maize egg cells, numerous smallvacuoles are located in the periphery of the cells and give no signal.To monitor total RNA distribution, embryo sac and nucellus cells werestained with acridine orange. Total RNA displays a similar pat-tern thanZmES1-4 transcripts and is uniformly distributed in the cytoplasm of thecells studied.

Example 4

The ZmES4 promoter is exclusively active in embryo sac cells oftransgenic maize lines As shown in FIG. 5, some 1.6 kbp upstream of thetranscription start point of ZmES4 is sufficient to drive acell-specific expression of the ZmES4::GFP fusion protein in the cellsof the female gametophyte (embryo sac). FIG. 5 b shows a signal of thefusion protein in the two synergids and very strong signals around thefiliform apparatus. CLSM analysis displays an expression also in the eggand cental cell, but the strongest signals were observed in the regionof the egg apparatus. All other cells/tissues of the ovary never showedany fluorescence of the GFP-fusion protein.

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1-23. (canceled)
 24. A protein obtained from the host cell containing avector comprising the nucleotide sequence selected from the groupconsisting of SEQ ID No. 4, 8 or 14, or a cell produced by theproliferation of a host cell of an egg cell from maize.
 25. The proteinhaving the activity of a molecule encoded by the sequences claim 24,which encodes a regulatory protein having the amino acid sequence of SEQID No.
 12. 26. An antibody or a fragment thereof, which is reactive withthe protein of claims 24 or
 25. 27. The antibody of claim 26 or thefragment thereof, wherein the antibody is a monoclonal antibody.
 28. Theantibody or the fragment thereof according to claim 26, wherein theantibody is a polyclonal antibody.
 29. The antibody or the fragmentthereof according to claim 26, wherein the antibody is a chimericantibody.
 30. The antibody or the fragment thereof according to claim29, wherein the chimeric antibody is composed of an antibody of ananimal and a lectin wherein the lectin is selected from the groupconsisting of plant lectins and animal lectins.
 31. The antibody or thefragment thereof according to claims 26, wherein the antibody or thefragment thereof has a detectable label.
 32. The antibody or thefragment thereof according to claims 26, wherein the antibody or thefragment thereof has undergone one or more modifications selected fromthe group consisting of, in reduction, oxidation and oligomerization.33. (canceled)
 34. A method for isolating embryo sac specific proteinsfrom a plant, whereby the antibody of claim 26 is used to screen and toisolate embryo sac specific proteins derived from the plant. 35-41.(canceled)
 42. A protein obtained from the host cell containing thevector comprising the nucleotide sequence comprising a sequence selectedfrom the group consisting of SEQ ID No. 4, 8 or 14, or a cell producedby the proliferation of the host cell,said host cell being an egg cellfrom maize, wherein the protein naturally occurs in Zea mays.
 43. Aprotein obtained according to the method of producing a proteinmodulating embryogenesis and endosperm development, wherein the hostcell containing a vector comprising the nucleotide sequence comprising asequence selected from the group consisting of SEQ ID No. 4, 8 or 14, iscultivated in culture medium under conditions allowing the synthesis ofthe protein, and the protein is obtained from the cultivated cells, theculture medium, or both, and wherein the protein naturally occurs in Zeamays.
 44. The antibody or the fragment thereof, wherein the antibody isa chimeric antibody, which is reactive with the protein of claims 24 or25, wherein the chimeric antibody comprises at least a portion of anantibody and at least a portion of a lectin, and wherein the lectin isselected from the group consisting of plant lectins and animal lectins.45. A plant demonstrating modified embryogenesis and/or modifiedendosperm development, wherein the method of production of the plantcomprises using at least one of the following: (i) a nucleotide sequencecomprising a sequence selected from the group consisting of SEQ ID No.4, 8 or 14, or the sequence being a DNA, cDNA or RNA molecule; (ii) avector comprising the nucleotide sequence selected from the groupconsisting of SEQ ID No. 4, 8 or 14, or a bacterial or viral vector,(iii) a protein obtained from the host cell containing the vector of(ii) or a cell produced by the profialtion of the host cell, or havingthe activity of a molecule encoded by the sequences defined in any oneof SEQ ID No. 4, 8, or 14, which encodes a regulatory protein thatparticipates in modulating embryogenesis and endosperm development; and(iv) the antibodies according to any one of claims 26-32.